Our laboratory is studying the 70-kDa class of heat shock proteins which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes the DnaJ class of proteins appears to be involved; this class of proteins acts as cofactors to the hsc70 class of proteins apparently "presenting" protein substrates to hsc70. However, the mechanism of this presentation is unknown. We have taken several approaches to understanding the mechanism of action of these proteins. First, we are carrying out a detailed investigation of the mechanism of action of hsc70 in uncoating bovine brain clathrin-coated vesicles. In carrying out this study we discovered that the protein cofactor, auxilin, is required for the uncoating process to occur, and that auxilin acts catalytically to induce clathrin baskets to bind to hsc70 in ATP. Therefore, auxilin appears to be acting like a DnaJ homolog and indeed it has a DnaJ region at its carboxyl-terminal end. We are also studying the direct effect of DnaJ homologs on hsc70. Our results show that several DnaJ homologs cause both an initial burst of ATP hydrolysis by hsc70 and reversible polymerization of the hsc70 in the presence of ATP. This may represent presentation of one hsc70 to another by the DnaJ under conditions where substrates are not present. We are currently investigating whether auxilin also directly interacts with hsc70 and causes its polymerization in the absence of clathrin baskets. We are also investigating the role of auxilin in the unusual observation that hsc70 supports only one round of uncoating before its activity is strongly inhibited in an unknown manner. In addition to these studies on uncoating, we have expressed a number of recombinant hsc70 mutants which bind either ATP or both ATP and ADP several orders of magnitude more weakly than normal hsc70. They also no longer hydrolyze ATP. These mutations had almost no effect on the properties of nucleotide-free hsc70; the mutated hsc70s still bound both clathrin triskelions and various peptide substrates. However, in contrast to the effects of strongly bound ATP and ADP, the weakly bound nucleotides had no effect on the binding of clathrin and peptides to hsc70. Furthermore, both polymerization by DnaJ homologs and auxilin dependent uncoating of clathrin were completely inhibited by the mutations suggesting that actions of hsc70 involving DnaJ homologs require strongly bound ATP and possibly ATP hydrolysis as well. We are currently transfecting hsc70 mutants, altered auxilin, and DnaJ homologs into mammalian cells to determine their effect on the actions of hsc70 in vivo.